Identification of Long Noncoding RNAs Expression Profiles Between Gallstone and Gallbladder Cancer Using Next-Generation Sequencing Analysis.

Background: Gallstone disease (GS) is an important risk factor for Gallbladder cancer (GBC). However, the mechanisms of the progression of GS to GBC remain unclear. Long non-coding RNA (lncRNA), modulates DNA/RNA/proteins at epigenetic, pre-transcriptional, transcriptional and posttranscriptional levels, and plays a potential therapeutic role in various diseases. This study aims to identify lncRNAs that have a potential impact on GS-promoted GBC progression. Methods and Results: Six GBC patients without GS, six normal gallbladder tissues, nine gallstones and nine GBC patients with GS were admitted to our hospital. The next-generation RNA-sequencing was performed to analyze differentially expressed (DE) lncRNA and messenger RNA (mRNA) in four groups. Then overlapping and specific molecular signatures were analyzed. We identified 29 co-DEGs and 500 co-DElncRNAs related to gallstone or GBC. The intersection and concatenation of co-DEGs and co-DElncRNA functionally involved in focal adhesion, Transcriptional misregulation in cancers, Protein digestion and absorption, and ECM-receptor interaction signaling pathways may contribute to the development of gallbladder cancer. Further exploration is necessary for early diagnosis and the potential treatment of GBC. FXYD2, MPZL1 and PAH were observed in both co-DEGs and co-DElncRNA and validated by qRT-PCR. Conclusion: Our data identified a series of DEGs and DElncRNAs, which were involved in the progression of GBC and GS-related metabolism pathways. Compared to GBC, the GS profile was more similar to para-tumor tissues in transcriptome level and lower risk of cancer. Further exploration is necessary from GBC patients with different periods of follow-up gallstone.

Open hepatic artery flow with portal vein clamping protects against bile duct injury compared to pringles maneuver.

BACKGROUND: Conventional hepatic artery and portal vein clamping strategies can prevent blood loss and ischemia-reperfusion liver injury, and such preventative measures are the key to successful liver surgery. However, ischemic-induced damage to cholangiocytes is rarely considered. Here, we aimed to investigate the effect of different hepatic inflow interruption methods on bile duct injury. METHODS: Forty rats were randomly grouped as sham, Pringle maneuver (PM) and hepatic arterial blood flow open (HAFO) groups. We evaluated liver histology and function in liver sections, and biliary histology, cholangiocyte apoptosis and proliferation, cytokine production, and bile composition. RNA sequencing is performed to explore possible molecular mechanisms. The Blood-biliary barrier permeability and tight junctions were analyzed by HRP injection, immunofluorescence staining and analysis of ZO-1 expression by immunoblotting. RESULTS: HAFO significantly attenuated ischemia-induced liver injury and decreased ALT, ALP, TBIL, and DBIL levels in serum. The histopathological observations showed that bile duct injury in the PM group was more serious than that in the HAFO group. The numbers of apoptotic biliary epithelial cells in HAFO-treated rat bile ducta were lower than those in the PM group. RNA-seq showed that tight junctions may be related to the mechanism underlying the protective effect of HAFO, as shown by the reduced HRP levels and increased ZO-1 and claudin-1/3 expression in the HAFO group compared to the PM group. CONCLUSION: Compared with PM, HAFO alleviated the ischemic injury to the biliary system, which was characterized by decreased biliary epithelial cell apoptosis, reduced inflammatory responses and decreased blood-biliary-barrier permeability.

Effect of nicotine on cholesterol gallstone formation in C57BL/6J mice fed on a lithogenic diet.

Gallstones are diseases of the biliary system caused by cholesterol supersaturation and/or deficiency in bile salts in bile. Early studies have shown that symptomatic gallstones are primarily a disease of non-smokers, raising the possibility that nicotine can prevent gallstone formation. The present study investigated the effect of nicotine on the formation of cholesterol gallstone in C57BL/6J mice. C57BL/6J mice (eight-weeks-old) were fed a normal or lithogenic diet (basic feed 82.45%, fat 15.8%, cholesterol 1.25% and sodium cholate 0.5%) and divided into five groups: normal diet (ND); ND + high dose nicotine (H); lithogenic diet (LD); LD + low dose nicotine (L) and LD + nicotine (H). They were treated with or without nicotine injection for 10 weeks. Nicotine treatment did not change the rate of cholesterol gallstone formation. There was no difference in TNFα, IL-1ß and IL-6 among the five groups. The LD group showed the highest cholesterol levels and there was significant suppression of the total cholesterol, low-density lipoprotein-cholesterol and total bile acid levels in the serum of the nicotine-treated mice. Quantitative PCR showed nicotine altered few bile acid metabolism-related genes expression in liver tissue and significantly altered cholesterol-metabolism genes in gallbladder tissue. Hematoxylin and eosin staining and western blotting showed that protein levels of farnesoid X receptor (FXR) and megalin in the gallbladder increased in the lithogenic-diet mice, which was significantly suppressed in the nicotine-treated mice. In vitro studies using gallbladder epithelial cells showed that chenodeoxycholic acids increased megalin expression, which could be attenuated by nicotine. Nicotine could regulate bile acid metabolism via the FXR-megalin/cubilin pathways, which potentially contribute to cholesterol nucleation and subsequent gallstone formation.

Nicotine-induced autophagy via AMPK/mTOR pathway exerts protective effect in colitis mouse model.

Epidemiological studies have shown that cigarette smoking is beneficial in ulcerative colitis and that nicotine may be responsible for this effect. However, the mechanism remains unclear. In a previous study, nicotine was found to induce autophagy in intestinal cells. Here, we evaluated the effect of nicotine-induced autophagy in a dextran sodium sulfate (DSS)-induced colitis mouse model. C57BL/6 adult male mice drank DSS water solution freely for seven consecutive days, and then tap water was administered. The effect of nicotine treatment was examined in the DSS model, including colon length, disease severity, histology of the colon tissue, and inflammation levels. Moreover, autophagy levels were detected by Western blot analysis (LC3II/LC3I, p62, and beclin-1). The levels of DSS-induced colitis were significantly decreased following nicotine treatment. The disease activity score, body weight, histologic damage scores, and the level of colonic inflammatory factors of nicotine-treated mice all decreased compared to those of the control mice. Additionally, nicotine enhanced the expression of LC3II/LC3I and beclin-1 but decreased the p62 protein level. Inhibiting autophagy by 3-MA attenuated the protective effects of nicotine on colitis. Additionally, both in vitro and in vivo experiments showed changes in AMPK-mTOR-P70S6K during this process. These results suggest that nicotine improved colitis by regulating autophagy and provided a protective effect against DSS-induced colitis.

hsa-miR-93 regulates MUCIN family gene expression via WNT/ß-catenin pathway in intrahepatic stone disease.

BACKGROUND: Mucin plays an essential role in the intrahepatic stone formation, but the mechanism of mucin regulation is unclear. OBJECTIVE: To investigate the potential implication of miR-93 and WNT pathway in the regulation of intrahepatic bile duct mucin expression. METHODS: Thirty patients with or without intrahepatic bile duct stones are involved; Reverse-transcription polymerase chain reaction was performed to evaluate the expression of MUC3, MUC4, MUC5B, MUC5AC mRNA and miR-93 levels. miR-NC or miR-93 mimics was transfected into intrahepatic biliary epithelial cells. Then mucins and Wnt pathway proteins were detected by the immunoblotting, and the target gene TCF7 were validated using the dual luciferase assay. ß-catenin, wnt4, and mucins were an immunohistochemical stain of the intrahepatic biliary epithelial tissues. RESULTS: The expression levels of MUC3, MUC4, MUC5B, and MUC5AC in patients with intrahepatic bile duct stones are higher than control, as well as Wnt pathway proteins (especially ß-catenin and wnt4). Mucins levels increased in wnt4, wnt5a or SB216763-treated HIBECs, and reduced by miR-93 mimics transfection. miR-93 directly targeted TCF7 and repressed Wnt pathway protein expression, which reversed the upregulation of mucin levels induced by wnt4 or wnt5a, but not SB216763. CONCLUSION: These results suggest a new potential mechanism in intrahepatic stones, regulating by miR-93/TCF7, non-canonical Wnt pathway, and mucins.

An integrated analysis of differential miRNA and mRNA expressions in human gallstones.

Gallstone disease, including cholesterol precipitation in bile, increased bile salt hydrophobicity and gallbladder inflammation. Here, we investigated miRNA and mRNA involved in the formation of gallstones, and explored the molecular mechanisms in the development of gallstones. Differentially expressed 17 miRNAs and 525 mRNA were identified based on Illumina sequencing from gallbladder mucosa of patients with or without gallstones, and were validated by randomly selected 6 miRNAs and 8 genes using quantitative RT-PCR. 114 miRNA target genes were identified, whose functions and regulating pathways were related to gallstones. The differentially expressed genes were enriched upon lipoprotein binding and some metabolic pathways, and differentially expressed miRNAs enriched upon ABC transportation and cancer related pathways. A molecular regulatory network consisting of 17 differentially expressed miRNAs, inclusive of their target genes, was constructed. miR-210 and its potential target gene ATP11A were found to be differentially expressed in both miRNA and mRNA profiles. ATP11A was a direct target of miR-210, which was predicted to regulate the ABC-transporters pathway. The expression levels of ATP11A in the gallstone showed inverse correlation with miR-210 expression, and up-regulation of miR-210 could reduce ATP11A expression in HGBEC. This is the first report that indicates the existence of differences in miRNA and mRNA expression in patients with or without gallstones. Our data shed light on further investigating the mechanisms of gallstone formation.